LOCUS       sequence-227778-        6358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GfaABC1D-iBARK-p2A-mCherry: Viral expression vector for astrocyte 
            Gaq silencing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6358)
  AUTHORS   Nagai J, Bellafard A, Qu Z, Yu X, Ollivier M, Gangwani MR, 
            Diaz-Castro B, Coppola G, Schumacher SM, Golshani P, Gradinaru V, 
            Khakh BS
  TITLE     Specific and behaviorally consequential astrocyte Gq GPCR signaling 
            attenuation in vivo with ibetaARK.
  JOURNAL   Neuron. 2021 Jul 21;109(14):2256-2274.e9. doi: 
            10.1016/j.neuron.2021.05.023. Epub 2021 Jun 16.
  PUBMED    34139149
REFERENCE   2  (bases 1 to 6358)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neuron.2021.05.023"; journalName: "Neuron"; date: 
            "2021-07-21- 21"; volume: "109"; issue: "14"; pages: "2256-2274.e9"
COMMENT     Sequence Label: GfaABC1D BARK p2A mCherry
FEATURES             Location/Qualifiers
     source          1..6358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     210..229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    597..618
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(633..638,639..656,657..663)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    671..687
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     676..698
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     695..711
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     695..711
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   749..878
                     /label=AAV2 ITR
     intron          1729..1861
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1844..1863
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1906..1924
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1961..1987
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1988..2011
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2444..2500
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2501..3211
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2646..2664)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2914..2933
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3236..3824
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3289..3309)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3707..3718)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3873..3994
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3884..3903)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3938..3957
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4156..4285
                     /label=AAV2 ITR
     primer_bind     complement(4304..4321)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4304..4320)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4313..4335)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4462..4917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4549..4568)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4759..4780
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4943..5047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5048..5116,5117..5908)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      join(6079..6358,1..309)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
       61 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
      121 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
      181 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
      241 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
      301 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
      361 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
      421 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
      481 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
      541 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      601 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      661 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
      721 gccagattta attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg
      781 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
      841 cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc
      901 atgctactta tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag
      961 gggacgctgc tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg
     1021 cagacagcca ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg
     1081 gcctcctctt catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg
     1141 cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt
     1201 gagcaggggg cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc
     1261 acagccccct aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct
     1321 attcagcctg tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca
     1381 gggggggaga ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga
     1441 gagctctccc catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt
     1501 gttgccaggg gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc
     1561 ccaggagcga gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca
     1621 agctttattg cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca
     1681 acagtctcga acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag
     1741 gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc
     1801 ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca
     1861 ggtgtccact cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta
     1921 taggctagcc tcgagctcaa gcttcgaatt caccaccatg tacccatacg atgttcctga
     1981 ctatgcggac tacaaagacg atgacgacaa gaagtacctg gaggaccggg gcgaggtgac
     2041 ttttgagaag atcttctccc agaagctggg gtacctgctt ttccgagact tctgcctgaa
     2101 gcacctggag gaggccaagc ccttggtaga gttctacgag gagatcaaga aatacgagaa
     2161 gctggagaca gaggaggagc gcctggtctg cagccgagag atcttcgaca cgtacatcat
     2221 gaaggagctg ctggcctgct cacatccttt ctcgaagagc gccattgagc acgtccaggg
     2281 ccatctggtg aagaagcagg tgcctccgga tctcttccag ccatatattg aagaaatttg
     2341 ccagaacctc cgaggagacg tgttccagaa attcatcgag agcgataaat tcacacgatt
     2401 ttgccagtgg aagaatgtag agctcaacat ccacggtagt ggcgcaacaa acttctctct
     2461 gctgaaacaa gccggagatg tcgaagagaa tcctggaccg atggtgagca agggcgagga
     2521 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     2581 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     2641 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     2701 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     2761 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     2821 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     2881 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     2941 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     3001 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     3061 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     3121 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     3181 caccggcggc atggacgagc tgtacaagta gaccggtatc atcaagctta tcgataatca
     3241 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3301 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3361 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     3421 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3481 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3541 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3601 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta
     3661 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3721 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3781 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat acctctagag
     3841 tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     3901 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3961 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     4021 gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     4081 tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt
     4141 taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4201 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4261 cctcagtgag cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac
     4321 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4381 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4441 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4501 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4561 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4621 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4681 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4741 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4801 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4861 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     4921 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4981 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5041 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5101 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5161 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5221 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5281 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5341 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5401 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5461 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5521 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5581 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5641 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5701 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5761 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5821 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5881 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5941 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6001 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6061 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6121 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6181 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6241 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6301 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactca
//
